Structural studies of ß-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus.

ß-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Šresolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (ß/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of ß-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.

Biochemical and Molecular Dynamics Study of a Novel GH 43 α-l-Arabinofuranosidase/ß-Xylosidase From Caldicellulosiruptor saccharolyticus DSM8903.

The complete hydrolysis of xylan can be facilitated by the coordinated action of xylanase and other de-branching enzymes. Here, a GH43 α-l-arabinofuranosidase/ß-xylosidase (CAX43) from Caldicellulosiruptor saccharolyticus was cloned, sequenced, and biochemically investigated. The interaction of the enzyme with various substrates was also studied. With a half-life of 120 h at 70°C, the produced protein performed maximum activity at pH 6.0 and 70°C. The enzyme demonstrated a higher activity (271.062 ± 4.83 U/mg) against para nitrophenol (pNP) α-L-arabinofuranosides. With xylanase (XynA), the enzyme had a higher degree of synergy (2.30) in a molar ratio of 10:10 (nM). The interaction of the enzyme with three substrates, pNP α-L-arabinofuranosides, pNP ß-D-xylopyranosides, and sugar beet arabinan, was investigated using protein modeling, molecular docking, and molecular dynamics (MD) simulation. During the simulation time, the root mean square deviation (RMSD) of the enzyme was below 2.5 Å, demonstrating structural stability. Six, five, and seven binding-interacting residues were confirmed against pNP α-L-arabinofuranosides, pNP ß-D-xylopyranosides, and arabinan, respectively, in molecular docking experiments. This biochemical and in silico study gives a new window for understanding the GH43 family's structural stability and substrate recognition, potentially leading to biological insights and rational enzyme engineering for a new generation of enzymes that perform better and have greater biorefinery utilization.

Garden and food waste co-fermentation for biohydrogen and biomethane production in a two-step hyperthermophilic-mesophilic process.

Co-fermentation of garden waste (GW) and food waste (FW) was assessed in a two-stage process coupling hyperthermophilic dark-fermentation and mesophilic anaerobic digestion (AD). In the first stage, biohydrogen production from individual substrates was tested at different volatile solids (VS) concentrations, using a pure culture of Caldicellulosiruptor saccharolyticus as inoculum. FW concentrations (in VS) above 2.9 g L-1 caused a lag phase of 5 days on biohydrogen production. No lag phase was observed for GW concentrations up to 25.6 g L-1. In the co-fermentation experiments, the highest hydrogen yield (46 ±â€¯1 L kg-1) was achieved for GW:FW 90:10% (w/w). In the second stage, a biomethane yield of 682 ±â€¯14 L kg-1 was obtained using the end-products of GW:FW 90:10% co-fermentation. The energy generation predictable from co-fermentation and AD of GW:FW 90:10% is 0.5 MJ kg-1 and 24.4 MJ kg-1, respectively, which represents an interesting alternative for valorisation of wastes produced locally in communities.

A non-linear model of hydrogen production by Caldicellulosiruptor saccharolyticus for diauxic-like consumption of lignocellulosic sugar mixtures.

BACKGROUND: Caldicellulosiruptor saccharolyticus is an attractive hydrogen producer suitable for growth on various lignocellulosic substrates. The aim of this study was to quantify uptake of pentose and hexose monosaccharides in an industrial substrate and to present a kinetic growth model of C. saccharolyticus that includes sugar uptake on defined and industrial media. The model is based on Monod and Hill kinetics extended with gas-to-liquid mass transfer and a cybernetic approach to describe diauxic-like growth. RESULTS: Mathematical expressions were developed to describe hydrogen production by C. saccharolyticus consuming glucose, xylose, and arabinose. The model parameters were calibrated against batch fermentation data. The experimental data included four different cases: glucose, xylose, sugar mixture, and wheat straw hydrolysate (WSH) fermentations. The fermentations were performed without yeast extract. The substrate uptake rate of C. saccharolyticus on single sugar-defined media was higher on glucose compared to xylose. In contrast, in the defined sugar mixture and WSH, the pentoses were consumed faster than glucose. Subsequently, the cultures entered a lag phase when all pentoses were consumed after which glucose uptake rate increased. This phenomenon suggested a diauxic-like behavior as was deduced from the successive appearance of two peaks in the hydrogen and carbon dioxide productivity. The observation could be described with a modified diauxic model including a second enzyme system with a higher affinity for glucose being expressed when pentose saccharides are consumed. This behavior was more pronounced when WSH was used as substrate. CONCLUSIONS: The previously observed co-consumption of glucose and pentoses with a preference for the latter was herein confirmed. However, once all pentoses were consumed, C. saccharolyticus most probably expressed another uptake system to account for the observed increased glucose uptake rate. This phenomenon could be quantitatively captured in a kinetic model of the entire diauxic-like growth process. Moreover, the observation indicates a regulation system that has fundamental research relevance, since pentose and glucose uptake in C. saccharolyticus has only been described with ABC transporters, whereas previously reported diauxic growth phenomena have been correlated mainly to PTS systems for sugar uptake.

Preparation and Characterization of Sugar-Assisted Cross-Linked Enzyme Aggregates (CLEAs) of Recombinant Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus ( CsCE).

High-efficiency lactulose-producing enzyme of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (WT- CsCE) was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Conditions for enzyme aggregation and cross-linking were optimized, and a sugar-assisted strategy with less damage to enzyme secondary structures was developed to improve the activity yield of CLEAs up to approximately 65%. The resulting CLEAs with multiple-layer network structures exhibited an enlarged optimal temperature range (70-80 °C) and maintained higher activity at 50-90 °C. Besides, CLEAs retained more than 95% of their initial activity after 10 successive batches at 60 °C, demonstrating superior reusability. Moreover, CLEAs displayed an equivalent or higher catalytic ability to free WT- CsCE in lactulose biosynthesis, and the final sugar ratios were similar, lactulose 58.8-61.7%, epilactose 9.3-10.2%, and lactose 27.8-30%, with a constant isomerization selectivity of 0.84-0.87 regardless of enzymes used and temperature applied. The proposed strategy is the first trial for enzymatic synthesis of lactulose catalyzed by CLEAs of WT- CsCE.

Investigation of Cr(VI) reduction potential and mechanism by Caldicellulosiruptor saccharolyticus under glucose fermentation condition.

This study examined the microbial reduction of hexavalent chromium [Cr(VI)] by an extremely thermophilic bacterium, Caldicellulosiruptor saccharolyticus, under glucose fermentation conditions at 70°C. Experimentation with different initial Cr(VI) concentrations confirmed that C. saccharolyticus had the ability to reduce Cr(VI) and immobilize Cr(III). At a concentration of 40mg/L, Cr(VI) was completely reduced within 12h, and 97% of the reduction product Cr(III) precipitated on the cell surface. Cr(VI) reduction was accelerated by the addition of neutral red (NR, an electron mediator), resulting in the reduction time shortened to 1h. The addition of CuCl2, a Ni-Fe hydrogenase inhibitor, also enhanced Cr(VI) reduction. Additionally, analysis of the relationship between Cr(VI) reduction and glucose fermentation suggested that different electron sources acted during CuCl2 and NR conditions. Hydrogen served as an electron donor under normal fermentation and NR conditions with the catalysis of Ni-Fe hydrogenase. However, when the activity of Ni-Fe hydrogenase was inhibited by CuCl2, C. saccharolyticus directly used reduction equivalents during glucose fermentation for intracellular Cr(VI) reduction. Therefore, our findings demonstrated high Cr(VI) reduction ability and different electron transfer pathways during Cr(VI) reduction by C. saccharolyticus.

Boosting dark fermentation with co-cultures of extreme thermophiles for biohythane production from garden waste.

Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS).

Biohythane production from marine macroalgae Sargassum sp. coupling dark fermentation and anaerobic digestion.

Potential biohythane production from Sargassum sp. was evaluated in a two stage process. In the first stage, hydrogen dark fermentation was performed by Caldicellulosiruptor saccharolyticus. Sargassum sp. concentrations (VS) of 2.5, 4.9 and 7.4gL(-1) and initial inoculum concentrations (CDW) of 0.04 and 0.09gL(-1) of C. saccharolyticus were used in substrate/inoculum ratios ranging from 28 to 123. The end products from hydrogen production process were subsequently used for biogas production. The highest hydrogen and methane production yields, 91.3±3.3Lkg(-1) and 541±10Lkg(-1), respectively, were achieved with 2.5gL(-1) of Sargassum sp. (VS) and 0.09gL(-1)of inoculum (CDW). The biogas produced contained 14-20% of hydrogen. Potential energy production from Sargassum sp. in two stage process was estimated in 242GJha(-1)yr(-1). A maximum energy supply of 600EJyr(-1) could be obtained from the ocean potential area for macroalgae production.

Biofilm formation by designed co-cultures of Caldicellulosiruptor species as a means to improve hydrogen productivity.

BACKGROUND: Caldicellulosiruptor species have gained a reputation as being among the best microorganisms to produce hydrogen (H2) due to possession of a combination of appropriate features. However, due to their low volumetric H2 productivities (Q H2), Caldicellulosiruptor species cannot be considered for any viable biohydrogen production process yet. In this study, we evaluate biofilm forming potential of pure and co-cultures of Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor owensensis in continuously stirred tank reactors (CSTR) and up-flow anaerobic (UA) reactors. We also evaluate biofilms as a means to retain biomass in the reactor and its influence on Q H2. Moreover, we explore the factors influencing the formation of biofilm. RESULTS: Co-cultures of C. saccharolyticus and C. owensensis form substantially more biofilm than formed by C. owensensis alone. Biofilms improved substrate conversion in both of the reactor systems, but improved the Q H2 only in the UA reactor. When grown in the presence of each other's culture supernatant, both C. saccharolyticus and C. owensensis were positively influenced on their individual growth and H2 production. Unlike the CSTR, UA reactors allowed retention of C. saccharolyticus and C. owensensis when subjected to very high substrate loading rates. In the UA reactor, maximum Q H2 (approximately 20 mmol · L(-1) · h(-1)) was obtained only with granular sludge as the carrier material. In the CSTR, stirring negatively affected biofilm formation. Whereas, a clear correlation was observed between elevated (>40 µM) intracellular levels of the secondary messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and biofilm formation. CONCLUSIONS: In co-cultures C. saccharolyticus fortified the trade of biofilm formation by C. owensensis, which was mediated by elevated levels of c-di-GMP in C. owensensis. These biofilms were effective in retaining biomass of both species in the reactor and improving Q H2 in a UA reactor using granular sludge as the carrier material. This concept forms a basis for further optimizing the Q H2 at laboratory scale and beyond.

Adsorption-based immobilization of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase on Bacillus subtilis spores.

Nonrecombinant spore was examined as a novel immobilization support to adsorb enzymes. Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE), efficiently producing lactulose using lactose as a single substrate, was immobilized on Bacillus subtilis spores via adsorption. The immobilization process was optimized, and the properties of immobilized CsCE and the interactions between the enzyme and spores were also investigated. Under the optimized conditions (pH 4.5, temperature 4 °C, reaction time 2 H, and initial enzyme concentration 2.4 mg/mL), the maximum adsorbed amount of CsCE was 1.47 mg/10(11) spores, and the enzyme activity recovery was 79.4%. The spore-immobilized CsCE presented a higher pH and thermal stability than a free enzyme. Total desorption of the immobilized enzyme was only achieved by treatment with 1.0 M NaCl at pH 1.0, indicating a strong adsorption between CsCE and B. subtilis spores. Efficient binding may require a potent combination of electrostatic and hydrophobic interactions between spores and an enzyme. The immobilized CsCE was applied to produce 395 g/L lactulose after 4 H. Moreover, the spores could be regenerated and the spore-immobilized enzyme showed good reusability as it retained approximately 70% of its initial activity after eight recycles.

Evaluation of assimilatory sulphur metabolism in Caldicellulosiruptor saccharolyticus.

Caldicellulosiruptor saccharolyticus has gained reputation as being among the best microorganisms to produce H2 due to possession of various appropriate features. The quest to develop an inexpensive cultivation medium led to determine a possible replacement of the expensive component cysteine, i.e. sulphate. C. saccharolyticus assimilated sulphate successfully in absence of a reducing agent without releasing hydrogen sulphide. A complete set of genes coding for enzymes required for sulphate assimilation were found in the majority of Caldicellulosiruptor species including C. saccharolyticus. C. saccharolyticus displayed indifferent physiological behaviour to source of sulphur when grown under favourable conditions in continuous cultures. Increasing the usual concentration of sulphur in the feed medium increased substrate conversion. Choice of sulphur source did not affect the tolerance of C. saccharolyticus to high partial pressures of H2. Thus, sulphate can be a principle sulphur source in an economically viable and more sustainable biohydrogen process using C. saccharolyticus.

Profiling carbohydrate composition, biohydrogen capacity, and disease resistance in potato

Background: Potato (Solanum tuberosum) is one of the most important sources of carbohydrates in human diet. Because of its high carbohydrate levels it recently has also received attention in biohydrogen production. To exploit the natural variation of potato with respect to resistance to major diseases, carbohydrate levels and composition, and capacity for biohydrogen production we analyzed tubers of native, improved, and genetically modified potatoes, and two other tuberous species for their glucose, fructose, sucrose, and starch content. Results: High-starch potato varieties were evaluated for their potential for Caldicellulosiruptor saccharolyticus-mediated biohydrogen production with Desirée and Rosita varieties delivering the highest biohydrogen amounts. Native line Vega1 and improved line Yagana were both immune to two isolates (A291, A287) of Phytophthora infestans. Conclusions: Our data demonstrate that native potato varieties might have great potential for further improving the multifaceted use of potato in worldwide food and biohydrogen production.

Complete conversion of major protopanaxadiol ginsenosides to compound K by the combined use of α-L-arabinofuranosidase and ß-galactosidase from Caldicellulosiruptor saccharolyticus and ß-glucosidase from Sulfolobus acidocaldarius.

The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing ß-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the ß-D-glucopyranoside-hydrolyzing ß-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb2, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb2, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.